Methods for selecting competent oocytes and competent embryos with high potential for pregnancy outcome

ABSTRACT

The present invention relates to a method for selecting a competent oocyte or a competent embryo.

FIELD OF THE INVENTION

The present invention relates to a method for selecting a competentoocyte or a competent embryo.

BACKGROUND OF THE INVENTION

In assisted reproductive technology (ART), pregnancy and birth ratesfollowing in vitro fertilization (IVF) attempts remain low. Indeed, 2out of 3 IVF cycles fail to result in pregnancy (SART 2004) and morethan 8 out of 10 transferred embryos fail to implant (Kovalevsky andPatrizio, 2005). In addition, more than 50% of IVF-born babies are frommultiple gestations (Reddy et al., 2007). Preterm deliveries that resultfrom multiple pregnancies caused by ART are estimated to account forapproximately $890 million of U.S. health care costs annually (Bromerand Seli, 2008).

Subjective morphological parameters are still a primary criterion toselect healthy embryos used for in IVF and ICSI programs. However, suchcriteria do not truly predict the competence of an embryo. Many studieshave shown that a combination of several different morphologic criterialeads to more accurate embryo selection (Balaban and Urman, 2006; LaSala et al., 2008; Scott et al., 2000). Morphological criteria forembryo selection are assessed on the day of transfer, and areprincipally based on early embryonic cleavage (25-27 h postinsemination), the number and size of blastomeres on day two or daythree, fragmentation percentage and the presence of multi-nucleation inthe 4 or 8 cell stage (Fenwick et al., 2002).

However, a recent study has shown that the selection of oocytes forinsemination does not improve outcome of ART as compared to the transferof all available embryos, irrespective of their quality (La Sala et al.,2008). There is a need to identify viable embryos with the highestimplantation potential to increase IVF success rates, reduce the numberof embryos for fresh replacement and lower multiple pregnancy rates.

For all these reasons, several biomarkers for embryo selection arecurrently being investigated (Haouzi et al., 2008; Pearson, 2006). Asembryos that result in pregnancy differ in their metabolomic profilescompared to embryos that do not, some studies are trying to identify amolecular signature that can be detected by non-invasive evaluation ofthe embryo culture medium (Brison et al., 2004; Gardner et al., 2001;Sakkas and Gardner, 2005; Seli et al., 2007; Zhu et al., 2007).

Genomics are also providing vital knowledge of genetic and cellularfunction during embryonic development. (McKenzie et al., 2004) and(Feuerstein et al., 2007) have reported, that the expression of severalgenes in cumulus cells, such as cyclooxygenase 2 (COX2), was indicativeof oocyte and embryo quality. Gremlin 1 (GREM1), hyaluronic acidsynthase 2 (HAS2), steroidogenic acute regulatory protein (STAR),stearoyl-coenzyme A desaturase 1 and 5 (SCD1 and 5), amphiregulin (AREG)and pentraxin 3 (PTX3) have also been shown to be positively correlatedwith embryo quality (Zhang et al., 2005). More recently, the expressionof glutathione peroxidase 3 (GPX3), chemokine receptor 4 (CXCR4), cyclinD2 (CCND2) and catenin delta 1 (CTNND1) in human cumulus cells have beenshown to be inversely correlated with embryo quality, based onearly-cleavage rates during embryonic development (van Montfoort et al.,2008). But, despite the fact that early cleavage has been shown to be areliable biomarker for predicting pregnancy (Lundin et al., 2001; VanMontfoort et al., 2004; Yang et al., 2007), these gene expressionprofiles of cumulus cells have not been studied with respect topregnancy outcome.

(Assou et al., 2008) and (Fourar et al., 2008) have reported thatBCL2-like 11 (BCL2L11), CASP8 and FADD-like apoptosis regulator (CFLAR),matrix metallopeptidase 14 (MMP14), nuclear factor I/B (NFIB) andphosphoenolpyruvate carboxykinase 1 (PCK1) gene expression profiles wereassociated with a successful pregnancy.

These genes expression profiles are associated with pregnancy outcome ingeneral but specific oocyte or embryo qualities as capacity of an embryoto implant or to develop without arrest have not been studied.

SUMMARY OF THE INVENTION

The present invention relates to a method for selecting an oocyte thatwill produce, upon fertilization, a viable embryo with a highimplantation rate leading to pregnancy, comprising the step of measuringthe expression level of 10 genes in a cumulus cell surrounding saidoocyte;

wherein said genes are ATF3, SIAT6, PRKACA, PLA2G5, GPC6, G0S2, RBMS1,NFIC, SLC40A1 and WNT6 and

wherein the oocyte is selected if said cumulus cell does not overexpressany of said 10 genes.

The present invention also relates to a method for selecting an embryowith a high implantation rate leading to pregnancy, comprising the stepof measuring the expression level of 10 genes in a cumulus cellsurrounding the embryo, wherein said genes are ATF3, SIAT6, PRKACA,PLA2G5, GPC6, G0S2, RBMS1, NFIC, SLC40A1 and WNT6 and wherein the embryois selected if said cumulus cell does not overexpress any of said 10genes.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined as set of 10 genes expressed in cumuluscells that are biomarkers for embryo potential and pregnancy outcome.They demonstrated that genes expression profile of cumulus cells whichsurrounds oocyte correlated to different pregnancy outcomes, allowingthe identification of a specific expression signature of embryosdeveloping toward pregnancy. Their results indicate that analysis ofcumulus cells surrounding the oocyte is a non-invasive approach forembryo selection.

Set of Predictive Genes

All the genes pertaining to the invention are known per se, and arelisted in the below Table A. Table A present the set of genes whosecombined expression profile has been shown to be informative forselecting an oocyte that will produce, upon fertilization, a viableembryo with a high implantation rate leading to pregnancy or forselecting a competent embryo with a high implantation potential leadingto pregnancy.

TABLE A set of predictive genes. Gene symbol Gene name Gene ID ATF3activating transcription factor 3 467 SIAT6 ST3 beta-galactosidealpha-2,3- 6487 sialyltransferase 3 PRKACA protein kinase,cAMP-dependent, 5566 catalytic, alpha PLA2G5 phospholipase A2, group V5322 GPC6 glypican 6 10082 G0S2 G0/G1switch 2 50486 RBMS1 RNA bindingmotif, single stranded 5937 interacting protein 1 NFIC nuclear factorI/C (CCAAT-binding 4782 transcription factor) SLC40A1 solute carrierfamily 40 (iron- 30061 regulated transporter), member 1 WNT6wingless-type MMTV integration site 7475 family, member 6

An object of the invention relates to a method for selecting an oocytethat will produce, upon fertilization, a viable embryo with a highimplantation rate leading to pregnancy, comprising the step of measuringthe expression level of 10 genes in a cumulus cell surrounding saidoocyte;

-   -   wherein said genes are ATF3, SIAT6, PRKACA, PLA2G5, GPC6, G0S2,        RBMS1, NFIC, SLC40A1 and WNT6 and    -   wherein the oocyte is selected if said cumulus cell does not        overexpress any of said 10 genes.

The methods of the invention may further comprise a step consisting ofcomparing the expression level of the genes in the sample with acontrol, wherein detecting differential in the expression level of thegenes between the sample and the control is indicative whether theoocyte produces, upon fertilization, a viable embryo with a highimplantation rate leading to pregnancy or the embryo is with a highimplantation rate leading to pregnancy.

The control may consist in sample comprising cumulus cells associatedwith a competent oocyte or in a sample comprising cumulus cellsassociated with an unfertilized oocyte or a non competent oocyte.

Preferably, the control consists in sample comprising cumulus cellsassociated with a competent oocyte.

A used herein the term “competent oocyte” refers to a female gamete oregg that when fertilized produces a viable embryo with a highimplantation rate leading to pregnancy.

According to the invention, the oocyte may result from a natural cycle,a modified natural cycle or a stimulated cycle for cTVF or TCSI. Theterm “natural cycle” refers to the natural cycle by which the female orwoman produces an oocyte. The term “modified natural cycle” refers tothe process by which, the female or woman produces an oocyte or twounder a mild ovarian stimulation with GnRH antagonists associated withrecombinant FSH or hMG. The term “stimulated cycle” refers to theprocess by which a female or a woman produces one ore more oocytes understimulation with GnRH agonists or antagonists associated withrecombinant FSH or hMG.

The term “cumulus cell” refers to a cell comprised in a mass of cellsthat surrounds an oocyte. These cells are believed to be involved inproviding an oocyte some of its nutritional, energy and or otherrequirements that are necessary to yield a viable embryo uponfertilization.

The expression “a cumulus cell overexpressed a gene” means that thelevel of expression of said gene in the cumulus cell is higher than theexpression level of said gene in a control sample.

Typically, the level of expression of genes may be measured by varioustechniques such as DNA microarray, quantitative RT-PCR,semi-quantitative RT-PCR or by proteomic approaches (ELISA method,Western blots . . . ).

Typically, an overexpressed gene has a level of expression at least 1.5,at least 2, at least 2.5, at least 5, at least 7.5 or at least 10 timeshigher than the level of expression of said gene in a control sample.

Preferably, the level of expression is measured by quantitative RT-PCRand an overexpressed gene has a level of expression at least 1.5, atleast 2, or at least 2.5 times higher than the level of expression ofsaid gene in a control sample.

The control sample may be a cumulus cell associated with a competentoocyte or embryo.

The methods of the invention are applicable preferably to women but maybe applicable to other mammals (e.g., primates, dogs, cats, pigs, cows .. . ).

The methods of the invention are particularly suitable for assessing theefficacy of an in vitro fertilization treatment. Accordingly theinvention also relates to a method for assessing the efficacy of acontrolled ovarian hyperstimulation (COS) protocol in a female subjectcomprising:

i) providing from said female subject at least one oocyte with itscumulus cells;

ii) determining by a method of the invention whether said oocyte is anoocyte that will produce, upon fertilization, a viable embryo with ahigh implantation rate leading to pregnancy.

Then after such a method, the embryologist may select the oocytes thatwill produce, upon fertilization, a viable embryo with a highimplantation rate leading to pregnancys and in vitro fertilized themthrough a classical in vitro fertilization (cIVF) protocol or under anintracytoplasmic sperm injection (ICSI) protocol.

A further object of the invention relates to a method for monitoring theefficacy of a controlled ovarian hyperstimulation (COS) protocolcomprising:

i) isolating from said woman at least one oocyte with its cumulus cellsunder natural, modified or stimulated cycles;

ii) determining by a method of the invention whether said oocyte is anoocyte that will produce, upon fertilization, a viable embryo with ahigh implantation rate leading to pregnancy;

iii) and monitoring the efficacy of COS treatment based on whether itresults in an oocyte that will produce, upon fertilization, a viableembryo with a high implantation rate leading to pregnancy.

The COS treatment may be based on at least one active ingredientselected from the group consisting of GnRH agonists or antagonistsassociated with recombinant FSH or hMG.

The present invention also relates to a method for selecting an embryowith a high implantation rate leading to pregnancy, comprising a step ofmeasuring the expression level of 10 genes in a cumulus cell surroundingthe embryo, wherein said genes are ATF3, SIAT6, PRKACA, PLA2G5, GPC6,G0S2, RBMS1, NFIC, SLC40A1 and WNT6 and wherein the embryo is selectedif said cumulus cell does not overexpress any of said 10 genes.

The term “embryo” refers to a fertilized oocyte or zygote. Saidfertilization may intervene under a classical in vitro fertilization(cIVF) or under an intracytoplasmic sperm injection (ICSI) protocol.

The term “classical in vitro fertilization” or “cIVF” refers to aprocess by which oocytes are fertilised by sperm outside of the body, invitro. IVF is a major treatment in infertility when in vivo conceptionhas failed. The term “intracytoplasmic sperm injection” or “ICSI” refersto an in vitro fertilization procedure in which a single sperm isinjected directly into an oocyte. This procedure is most commonly usedto overcome male infertility factors, although it may also be used whereoocytes cannot easily be penetrated by sperm, and occasionally as amethod of in vitro fertilization, especially that associated with spermdonation.

The term “competent embryo” refers to an embryo with a high implantationrate leading to pregnancy. The term “high implantation rate” means thepotential of the embryo when transferred in uterus, to be implanted inthe uterine environment and to give rise to a viable foetus, which inturn develops into a viable offspring absent a procedure or event thatterminates said pregnancy.

The methods of the invention may further comprise a step consisting ofcomparing the expression level of the genes in the sample with acontrol, wherein detecting differential in the expression level of thegenes between the sample and the control is indicative whether theembryo is competent.

The control may consist in sample comprising cumulus cells associatedwith an embryo that gives rise to a viable foetus or in a samplecomprising cumulus cells associated with an embryo that does not giverise to a viable foetus.

Preferably, the control consists in sample comprising cumulus cellsassociated with an embryo that gives rise to a viable foetus.

It is to note that the methods of the invention leads to an independencefrom morphological considerations of the embryo. Two embryos may havethe same morphological aspects but by a method of the invention maypresent a different implantation rate leading to pregnancy.

The methods of the invention are applicable preferably to women but maybe applicable to other mammals (e.g. primates, dogs, cats, pigs, cows .. . ).

The present invention also relates to a method for determining whetheran embryo is an embryo with a high implantation rate leading topregnancy, comprising a step consisting in measuring the expressionlevel of 10 genes in a cumulus cell surrounding the embryo, wherein saidgenes are ATF3, SIAT6, PRKACA, PLA2G5, GPC6, G0S2, RBMS1, NFIC, SLC40A1and WNT6.

The present invention also relates to a method for determining whetheran embryo is an embryo with a high implantation rate leading topregnancy, comprising:

i) providing an oocyte with its cumulus cells

ii) in vitro fertilizing said oocyte

iii) determining whether the embryo that results from step ii) iscompetent by determining by a method of the invention whether saidoocyte of step i), is an oocyte that will produce, upon fertilization, aviable embryo with a high implantation rate leading to pregnancy.

The methods of the invention are particularly suitable for enhancing thepregnancy outcome of a female. Accordingly the invention also relates toa method for enhancing the pregnancy outcome of a female comprising:

i) selecting an embryo with a high implantation rate leading topregnancy by performing a method of the invention

iii) implanting the embryo selected at step i) in the uterus of saidfemale.

The method as above described will thus help embryologist to avoid thetransfer in uterus of embryos with a poor potential for pregnancy outcome.

The method as above described is also particularly suitable for avoidingmultiple pregnancies by selecting the competent embryo able to lead toan implantation and a pregnancy.

In all above cases, the methods described the relationship between genesexpression profile of cumulus cells and embryo and pregnancy outcomes.

Methods for determining the Expression Level of the Genes of theInvention:

Determination of the expression level of the genes as above described inTable A can be performed by a variety of techniques. Generally, theexpression level as determined is a relative expression level.

More preferably, the determination comprises contacting the sample withselective reagents such as probes, primers or ligands, and therebydetecting the presence, or measuring the amount, of polypeptide ornucleic acids of interest originally in the sample. Contacting may beperformed in any suitable device, such as a plate, microtiter dish, testtube, well, glass, column, and so forth. In specific embodiments, thecontacting is performed on a substrate coated with the reagent, such asa nucleic acid array or a specific ligand array. The substrate may be asolid or semi-solid substrate such as any suitable support comprisingglass, plastic, nylon, paper, metal, polymers and the like. Thesubstrate may be of various forms and sizes, such as a slide, amembrane, a bead, a column, a gel, etc. The contacting may be made underany condition suitable for a detectable complex, such as a nucleic acidhybrid or an antibody-antigen complex, to be formed between the reagentand the nucleic acids or polypeptides of the sample.

In a preferred embodiment, the expression level may be determined bydetermining the quantity of mRNA.

Methods for determining the quantity of mRNA are well known in the art.For example the nucleic acid contained in the samples (e.g., cell ortissue prepared from the patient) is first extracted according tostandard methods, for example using lytic enzymes or chemical solutionsor extracted by nucleic-acid-binding resins following the manufacturer'sinstructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR).Preferably quantitative or semi-quantitative RT-PCR is preferred.Real-time quantitative or semi-quantitative RT-PCR is particularlyadvantageous.

Other methods of Amplification include ligase chain reaction (LCR),transcription-mediated amplification (TMA), strand displacementamplification (SDA) and nucleic acid sequence based amplification(NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequencecomplementarity or homology to the mRNA of interest herein find utilityas hybridization probes or amplification primers. It is understood thatsuch nucleic acids need not be identical, but are typically at leastabout 80% identical to the homologous region of comparable size, morepreferably 85% identical and even more preferably 90-95% identical. Incertain embodiments, it will be advantageous to use nucleic acids incombination with appropriate means, such as a detectable label, fordetecting hybridization. A wide variety of appropriate indicators areknown in the art including, fluorescent, radioactive, enzymatic or otherligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to1000 nucleotides in length, for instance of between 10 and 800, morepreferably of between 15 and 700, typically of between 20 and 500.Primers typically are shorter single-stranded nucleic acids, of between10 to 25 nucleotides in length, designed to perfectly or almostperfectly match a nucleic acid of interest, to be amplified. The probesand primers are “specific” to the nucleic acids they hybridize to, i.e.they preferably hybridize under high stringency hybridization conditions(corresponding to the highest melting temperature Tm, e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification anddetection method may be assembled as a kit. Such a kit includesconsensus primers and molecular probes. A preferred kit also includesthe components necessary to determine if amplification has occurred. Thekit may also include, for example, PCR buffers and enzymes; positivecontrol sequences, reaction control primers; and instructions foramplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise thesteps of providing total RNAs extracted from cumulus cells andsubjecting the RNAs to amplification and hybridization to specificprobes, more particularly by means of a quantitative orsemi-quantitative RT-PCR.

In another preferred embodiment, the expression level is determined byDNA chip analysis. Such DNA chip or nucleic acid microarray consists ofdifferent nucleic acid probes that are chemically attached to asubstrate, which can be a microchip, a glass slide or amicrosphere-sized bead. A microchip may be constituted of polymers,plastics, resins, polysaccharides, silica or silica-based materials,carbon, metals, inorganic glasses, or nitrocellulose. Probes comprisenucleic acids such as cDNAs or oligonucleotides that may be about 10 toabout 60 base pairs. To determine the expression level, a sample from atest subject, optionally first subjected to a reverse transcription, islabelled and contacted with the microarray in hybridization conditions,leading to the formation of complexes between target nucleic acids thatare complementary to probe sequences attached to the microarray surface.The labelled hybridized complexes are then detected and can bequantified or semi-quantified. Labelling may be achieved by variousmethods, e.g. by using radioactive or fluorescent labelling. Manyvariants of the microarray hybridization technology are available to theman skilled in the art (see e.g. the review by Hoheisel, Nature Reviews,Genetics, 2006, 7:200-210).

In this context, the invention further provides a DNA chip comprising asolid support which carries nucleic acids that are specific to the geneslisted in table A.

Other methods for determining the expression level of said genes includethe determination of the quantity of proteins encoded by said genes.

Such methods comprise contacting the sample with a binding partnercapable of selectively interacting with a marker protein present in thesample. The binding partner is generally an antibody that may bepolyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standardelectrophoretic and immunodiagnostic techniques, including immunoassayssuch as competition, direct reaction, or sandwich type assays. Suchassays include, but are not limited to, Western blots; agglutinationtests; enzyme-labeled and mediated immunoassays, such as ELISAs;biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, etc. The reactions generally include revealinglabels such as fluorescent, chemiluminescent, radioactive, enzymaticlabels or dye molecules, or other methods for detecting the formation ofa complex between the antigen and the antibody or antibodies reactedtherewith.

The aforementioned assays generally involve separation of unboundprotein in a liquid phase from a solid phase support to whichantigen-antibody complexes are bound. Solid supports which can be usedin the practice of the invention include substrates such asnitrocellulose (e. g., in membrane or microtiter well form),polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

More particularly, an ELISA method can be used, wherein the wells of amicrotiter plate are coated with an antibody against the protein to betested. A biological sample containing or suspected of containing themarker protein is then added to the coated wells. After a period ofincubation sufficient to allow the formation of antibody-antigencomplexes, the plate (s) can be washed to remove unbound moieties and adetectably labeled secondary binding molecule added. The secondarybinding molecule is allowed to react with any captured sample markerprotein, the plate washed and the presence of the secondary bindingmolecule detected using methods well known in the art.

Alternatively an immunohistochemistry (IHC) method may be preferred. IHCspecifically provides a method of detecting targets in a sample ortissue specimen in situ. The overall cellular integrity of the sample ismaintained in IHC, thus allowing detection of both the presence andlocation of the targets of interest. Typically a sample is fixed withformalin, embedded in paraffin and cut into sections for staining andsubsequent inspection by light microscopy. Current methods of IHC useeither direct labeling or secondary antibody-based or hapten-basedlabeling. Examples of known IHC systems include, for example, EnVision™(DakoCytomation), Powervision® (Immunovision, Springdale, Ariz.), theNBA™ kit (Zymed Laboratories Inc., South San Francisco, Calif.),HistoFine® (Nichirei Corp, Tokyo, Japan).

In particular embodiment, a tissue section (e.g. a sample comprisingcumulus cells) may be mounted on a slide or other support afterincubation with antibodies directed against the proteins encoded by thegenes of interest. Then, microscopic inspections in the sample mountedon a suitable solid support may be performed. For the production ofphotomicrographs, sections comprising samples may be mounted on a glassslide or other planar support, to highlight by selective staining thepresence of the proteins of interest.

Therefore IHC samples may include, for instance: (a) preparationscomprising cumulus cells (b) fixed and embedded said cells and (c)detecting the proteins of interest in said cells samples. In someembodiments, an THC staining procedure may comprise steps such as:cutting and trimming tissue, fixation, dehydration, paraffininfiltration, cutting in thin sections, mounting onto glass slides,baking, deparaffination, rehydration, antigen retrieval, blocking steps,applying primary antibodies, washing, applying secondary antibodies(optionally coupled to a suitable detectable label), washing, counterstaining, and microscopic examination.

The invention also relates to a kit for performing the methods as abovedescribed, wherein said kit comprises means for measuring the expressionlevel the levels of the genes of Table A that are indicative whether theoocyte or the embryo is competent.

The invention will be further illustrated by the following FIGURES andexamples. However, these examples and FIGURES should not be interpretedin any way as limiting the scope of the present invention.

FIGURE

FIG. 1: Box plot of set of predictive genes.

For each gene, the relative intensities of embryo's cumulus cells thatgave pregnancy are shown at the left side and the relative intensitiesof those that did not give pregnancy at the right side.

EXAMPLE A Non-Invasive Test for Assessing Embryo Potential by GeneExpression Profiles of Human Cumulus Cells

Material & Methods:

Patients and IVF Treatment:

In this retrospective study, normo-responder patients (n=30) aged of30.9 years±2.5 and referred to our centre for ICSI (Intra CytoplasmicSperm Injection) for male infertility factor were studied. Patients werestimulated with a combination of GnRH agonist or antagonist withrecombinant FSH (GonalF, Puregon; respectively of Merck-Serono andOrganon) or with hMG (Menopur, Ferring). Ovarian response was evaluatedby serum estradiol level and ultrasound examination to monitor follicledevelopment Retrieval of oocytes was performed 36 hours after hCGadministration (5000 IU), under ultrasound guidance.

Assessment of Embryo Quality:

On day 2 and 3 postmicroinjection, the quality parameters ofindividually cultured embryo were evaluated using the number ofblastomeres and the degree of fragmentation as criteria (grade 1-2:equally sized blastomeres and 0-20% fragmentation, grade 3-4: no equallysized blastomeres and more than 20% fragmentation. A top-quality embryowas defined on day 3 as 6-8 cells, equally sized blastomeres and nofragmentation. One or two embryos were transferred on day 3 after oocyteretrieval. Clinical pregnancy was evaluated two and six weeks afterembryo transfer based respectively on serum Beta-hCG and ultrasoundexamination (presence of gestational sac with heart beat).

Cumulus Cells:

All cumulus cells (CC) samples were frozen on egg collection day. Then,one to 3 CC samples per patient were randomly selected for microarrayanalysis. A total of 50 CC samples were collected from 50 single oocytesand analyzed individually: 34 CC from grade 1-2 embryos (n=20 patients),11 CC from grade 3-4 embryos (n=10 patients) and 5 CC from unfertilizedoocytes (n=5 patients) (Table 1).

TABLE 1 The Characteristics of cumulus cells samples in this study 30patients 5 Patients 45 CC 5 CC G1/2 (34 CC) G3/4 (11 CC) cumulus cellsfrom P+ P− NT unfertilized oocyte (5CC) chips nbr 18 16 11 5 Patientsnbr 11 9 10 5 CC nbr 18 16 11 5

CC: cumulus cells, P+: cumulus cells from embryos with positivepregnancy outcome, P−: cumulus cells from embryos without pregnancyoutcome, G1/2 cumulus cells from grade 1-2 embryos, G3/4: cumulus cellsfrom grade 3-4 embryos, NT: no transfer.

The data analysis was performed under double blind conditions in whichpregnancy outcome was disclosed only after microarrays were hybridized.Regarding pregnancy outcome, the 45 CC from fertilized oocytes included16 CC from grade 1-2 embryos that did not result into pregnancy (n=9patients), 18 CC associated with a positive pregnancy outcome (n=11patients) and 11 CC from grade 3-4 embryos that were not transferred.Cumulus cells were stripped immediately following oocyte recovery (<40 hpost hCG administration). Cumulus cells were mechanically removed andwashed in culture medium and immediately frozen at −80° C. in RLT RNAextraction buffer (RNeasy kit, Qiagen, Valencia, Calif., USA) before RNAextraction.

Granulosa Cells:

An independent group of normo responder patients (n=8) (age 34.8years±3.2) referred for ICSI program for male infertility factor wasselected for granulosa cells collection (8 samples). Immediately afteroocyte recovery, follicular fluids from matures follicles (>17 mm) ofthe same patient were pooled, after removal of the cumulus oocytecomplex and diluted in ⅓ volume of HBSS solution (BioWhittaker) in 50 mlbatches, representing one sample. Granulosa cells purification wasadapted from the protocol by (Kolena et al., 1983). Following a 20 min.centrifugation at 500 g in swinging buckets, granulosa cells werecollected on a Ficoll cushion (12 ml Lymphocyte separation medium,BioWhittaker). They were successively washed in HBSS and PBS, incubated5 min. in blood lysis buffer (KHCO₃ 10 mM, NH₄Cl 150 mM, EDTA 0.1 mM) toremove red blood cells, counted and pelleted in PBS before lysis in RLTbuffer (Quiagen) and storage at −80° C. The number of follicularpuncture and the number of purified granulosa cells ranged from 6 to 12and from 2 10⁶ to 9 10⁶ respectively.

Complementary RNA (cRNA) Preparation and Microarray Hybridization:

CC and granulosa cells RNA was extracted using the micro RNeasy Kit(Qiagen). The total RNA quantity was measured with a Nanodrop ND-1000spectrophotometer (Nanodrop Technologies Inc., DE, USA) and RNAintegrity was assessed with an Agilent 2100 Bioanalyzer (Agilent, PaloAlto, Calif., USA). cRNA was prepared with two rounds of amplificationaccording to the manufacturer's protocol “double amplification”(Two-Cycle cDNA Synthesis Kit, Invitrogen) starting from total RNA(ranging from 70 ng to 100 ng). cRNA obtained after the firstamplification ranged from 0.1 μg/μl to 1.9 μg/μl and after the secondamplification ranged from 1.6 μg/μl to 4.5 μg/μl.

Labelled fragmented cRNA (12 μg) was hybridized to oligonucleotideprobes on an Affymetrix HG-U133 Plus 2.0 array containing 54 675 sets ofoligonucleotide probes (“probeset”) which correspond to ≈30 000 uniquehuman genes or predicted genes. Each cumulus and granulosa sample wasput individually on a microarray chip.

Data Processing:

Scanned GeneChip images were processed using Affymetrix GCOS 1.4software to obtain an intensity value and a detection call (present,marginal or absent) for each probeset, using the default analysissettings and global scaling as first normalization method, with atrimmed mean target intensity value (TGT) of each array arbitrarily setto 100. Probe intensities were derived using the MAS5.0 algorithm. Thisalgorithm also determines whether a gene is expressed with a definedconfidence level or not (“detection call”). This “call” can either be“present” (when the perfect match probes are significantly morehybridized than the mismatch probes, p-value <0.04), “marginal” (forp-values >0.04 and <0.06) or “absent” (p-value >0.06). The microarraydata were obtained in our laboratory in agreement with the MinimalInformation about a Microarray Experiment MIAME recommendations (Brazmaet al. 2001).

Data Analysis and Visualisation:

Significant Analysis of microarrays (SAM) (Tusher et al., 2001)(http://www-stat.stanford.edu/˜tibs/SAM/) was used to identify geneswhose expression varied significantly between sample groups. SAMprovides mean or median fold change values (FC) and a false discoveryrate (FDR) confidence percentage based on data permutation (mean foldchange >2 and FDR <5%). Array analysis allowing the comparison of geneexpression profile between cumulus cell samples and granulosa cellsamples is first based on the significant RNA detection (detection call“present” or “absent”) and then, submitted to a SAM (SignificantAnalysis of microarrays) to identify genes whose expression variedsignificantly between sample groups. To perform the comparison of geneexpression profile between cumulus cell samples according embryonicquality and/or pregnancy outcome, a non-supervised selection ofprobesets using a variation coefficient (CV ≧40%) and a AbsentPresent“detection call” filter was performed before the SAM. To compare profileexpression of cumulus cells from altered (grade 3-4) and good (grade1-2) embryonic development, or from embryos leading, or not, to apregnancy, we performed an unsupervised classification with bothprincipal component analysis (PCA) and hierarchical clustering (de Hoonet al., 2004; Eisen et al., 1998). The PCA involved original scriptsbased on the R statistics software through the RAGE web interface(http://rage.montp.inserm.fr) (Reme et al., 2008). Hierarchicalclustering analysis based on the expression levels of varying probeswere performed with the CLUSTER and TREEVIEW software packages. Touncover functional biological networks and top canonical pathways, weimported gene expression signatures into the Ingenuity Pathways Analysis(IPA) Software (Ingenuity Systems, Redwood City, Calif., USA).

Quantitative RT-PCR Analyses:

For qRT-PCR analysis, 10 CC samples used in the microarray experimentswere selected according to their pregnancy outcome (5 CC samplesassociated to a negative outcome and 5 to a positive outcomecorresponding to 10 patients). Labelled cRNA (1 μg) from the patient wasused to generate first strand cDNA. These cDNAs (5 μl of a 1/10dilution) were used for real-time quantitative PCR reactions accordingto the manufacturer's recommendations (Applied Biosytems). The 20 μlreaction mixture consisted of cDNA (5 μl), 1 μM of primers and 10 μl ofTaqman Universal PCR Master Mix (Applied Biosystem). The amplificationwas measured during 40 cycles with an annealing temperature at 60° C.The amount of PCR product produced in every cycle step of the PCRreaction is monitored by TaqMan probe. A threshold is set in theexponential phase of the amplification curve, from which the cyclenumber (“Ct” for “Cycle Threshold”) is read off. The Ct-value is used inthe calculation of relative mRNA transcript levels. Effectiveness (E) ofthe PCR was measured. This effectiveness is obtained by a standard curvecorresponding to the primers used. Quantitative reverse transcriptasepolymerase chain reaction (QRT-PCR) was performed using the ABI Prism7000 sequence detection system (Applied Biosystems) and normalized toPGK1 for each sample using the following formula: E_(tested primer)^(ΔCt)/E_(PGK1) ^(ΔCt) (E=10^(−1/slope)), ΔCt=Ct control−Ct unknown,control=one CC sample of the non-pregnant group). Each sample wasanalysed in duplicate, and multiple water blanks were included with theanalysis.

Results

Gene Expression Profile of CC According to Embryo Outcome:

To identify a gene expression profile in CC that correlated with embryooutcome, we established a gene expression signature for each outcomecategory: CC of unfertilized oocytes, CC from oocytes that resulted inembryo development but extensive fragmentation (grade 3-4), and CC fromoocytes that resulted in embryo development with no or limitedfragmentation (grade 1-2). Granulosa cells samples were taken as areference tissue (control). Indeed, granulosa cells are cells closelyrelated to CC as opposed to other adult tissues. The use of thisreference tissue lowered the number of differentially expressed genesrelated to crude lineage differences, thus facilitating theidentification of subtle variation in the CC/oocyte interplay. A SAManalysis showed that 2605 genes were upregulated in the unfertilizedgroup, 2739 in the grade 3/4 group and 2482 in the grade 1-2 group witha FDR <5%. Conversely, 4270, 4349 and 4483 genes, were downregulated,respectively. These lists of genes were then intersected to determinetheir overlap. While 449 up and 890 down expressed genes were in commonin all three groups, each category displayed a specific gene expressionprofile. Interestingly, 860 up-regulated genes, including for exampleGalanin and Gap Junction A5 (GJA5), and 1416 down-regulated genes,including HLA-G and EGR1 were specifically modulated in cumulus cellsassociated to a good morphological embryonic quality. It must be notedthat although the grade 1-2 group displayed a strong gene expressionprofile, this group was heterogeneous regarding to pregnancy outcome andincluded 18 CC samples associated with embryos that resulted inpregnancy (including 4 twin pregnancies) but also 16 CC samplesassociated with embryos that failed to give rise to pregnancy.

Gene Expression Profile of CC According to Pregnancy Outcome:

CC samples were therefore compared according to the pregnancy outcome. ASAM analysis delineated a “pregnancy outcome” list of 630 genes thatvaried significantly (FDR <5%) between the two group of patients(pregnancy versus no pregnancy). PCA and hierarchical clusteringconfirmed that this 630 gene list indeed segregated a majority of CCsamples associated with pregnancy from those associated with nopregnancy. Of note, genes from the “pregnancy outcome” list werepredominantly upregulated in samples associated with a good outcome. The“pregnancy outcome” expression signature was particularly marked in asub-group of 10 CC samples from embryos associated to the “pregnancy”group.

Functional Annotation of the Pregnancy Outcome Gene List:

To investigate biological processes correlated to embryo achievingpregnancy, Ingenuity and Pubmed databases were used to annotate the 630genes from the “pregnancy outcome” gene list. Among genes whoseoverexpression is associated with pregnancy, the most significantlyoverrepresented pathways were “oxidative stress”, “TR/RXR activation”,“G2/M transition of the cell cycle”, “xenobiotic metabolism” and“NFKappaB” signalling. Among these pathways, the most representativegenes were interleukins, chemokines, adptator proteins and kinases:IL1Beta (×4.5 in pregnancy samples versus no pregnancy, P=0.001), IL16(×4.8, P=0.001), IL8 (×2.6, P=0.007), IL1RN (×2.1, P=0.0051), IL17RC(×3.6, P=0.001), TIRAP (×8.0, P=0.001), CXCL12 (×3.1, P=0.001), CCR5(×2.6, P=0.0051), and PCK1 (×3.4, P=0.001). Strikingly, numerous genesinvolved in the regulation of apoptosis were significantly modulated inCC samples from oocytes resulting in a pregnancy. These genes wereBCL2L11 (×6.9, P<0.001), CRADD (×2, P=0.0036), NEMO (×4.6, P<0.001),BCL10 (×3.1, P=<0.001), SERPINB8 (×9.1, P<0.001). and TNFSFI3 (×2.5,P=0.0038).

On the other hand, genes associated with no pregnancy were correlatedwith the following pathways: G2/M DNA damage and checkpoint regulationof the cell cycle, “Sonic hedgehog”, “IGF-1”, “complement system” and“Wnt/Beta-catenin” signalling. Representative genes correlated with nopregnancy included NFIB (×0.3, P<0.001), MAD2L1 (×0.4, P<0.001) andIGF1R (×0.4, P<0.001).

SAM Analysis:

The SAM analysis of CC according to pregnancy outcome identified the 45genes of Table B that are biomarkers for embryo potential that woulddifferentiate between oocytes that produced embryos resulting in apregnancy versus those that did not result in pregnancy based on genesexpression of CC analysis. QRT-PCR was used to confirm independently themicroarray data. We analyzed the differential expression of 36up-regulaled genes and 9 down-regulated genes between CC from grade 1-2embryos did not achieve pregnancy and CC from grade 1-2 embryosachieving pregnancy.

TABLE B set of predictive genes Gene Symbol Gene name Gene ID WNT6wingless-type MMTV integration site family, 7475 member 6 LRCH4leucine-rich repeats and calponin homology 4034 (CH) domain containingPAX8 paired box 8 7849 CABP4 calcium binding protein 4 57010 PDE5Aphosphodiesterase 5A, cGMP-specific 8654 BCL2L11 BCL2-like 11 (apoptosisfacilitator) 10018 PCK1 phosphoenolpyruvate carboxykinase 1 (soluble)5105 TCF20 transcription factor 20 (AR1) 6942 SLAMF6 SLAM family member6 114836 EPOR erythropoietin receptor 2057 CACNG6 calcium channel,voltage-dependent, gamma 59285 subunit 6 NLRP1 NLR family, pyrin domaincontaining 1 22861 PECAM1 platelet/endothelial cell adhesion molecule5175 NOS1 nitric oxide synthase 1 (neuronal) 4842 ATF3 Activatingtranscription factor 3 467 KRTAP8 keratin associated protein 8-1 337879GRIK5 Glutamate receptor, ionotropic, kainate 5 2901 SLC24A3 solutecarrier family 24 57419 (sodium/potassium/calcium exchanger), member 3SLC5A12 solute carrier family 5 (sodium/glucose 159963 cotransporter),member 12 SLCA10A2 Solute carrier family 10 (sodium/bile acid 6555cotransporter family), member 2 SLCO1A2 solute carrier organic aniontransporter family, 6579 member 1A2 SLC25A5 solute carrier family 25(mitochondrial carrier; 292 adenine nucleotide translocator), member 5MG29 or synaptophysin-like 2 284612 SYPL2 NLGN2 neuroligin 2 57555PRKACA protein kinase, cAMP-dependent, catalytic, 5566 alpha FOSB FBJmurine osteosarcoma viral oncogene 2354 homolog B SIAT6 ST3beta-galactoside alpha-2,3- 6487 sialyltransferase 3 LOXL2 lysyloxidase-like 2 4017 PRF1 perforin 1 (pore forming protein) 5551 ADPRHADP-ribosylarginine hydrolase 141 APBB3 amyloid beta (A4) precursorprotein-binding, 10307 family B, member 3 EGR3 early growth response 31960 CNR2 cannabinoid receptor 2 (macrophage) 1269 IFITM1 Interferoninduced transmembrane protein 1 (9- 8519 27) PLA2G5 phospholipase A2,group V 5322 CAMTA1 calmodulin binding transcription activator 1 23261SOX4 SRY (sex determining region Y)-box 4 6659 NFIB nuclear factor I/B4781 NFIC nuclear factor I/C (CCAAT-binding 4782 transcription factor)RBMS1 RNA binding motif, single stranded interacting 5937 protein 1 G0S2G0/G1switch 2 50486 FAT3 FAT tumor suppressor homolog 3 (Drosophila)120114 SLC40A1 solute carrier family 40 (iron-regulated 30061transporter), member 1 GPC6 glypican 6 10082 IGF1R insulin-like growthfactor 1 receptor 3480

Reliability Test of the Candidate Genes and Correlation Between CC GenesExpression Profile and Absence of Pregnancy

In order to test the reliability of the 45 gene list, a prospectivestudy was conducted including young (<36 years) normal responderpatients referred to our centre for ICSI for male infertility. Theembryo selection occurred either according to the gene expressionprofile in CCs (group 1) or to morphological aspects (group 2 used ascontrol). For each group, two embryos were replaced. For the first 60patients (30 patients/group), on egg collection day, in group 1, each CCsample was collected individually and processed for gene expressionanalysis. CC samples (n=267) were analyzed.

Quantitative RT-PCR analysis was performed to measure the relativeabundance of the transcripts of interest genes in CCs, and expressiondata for all biomarkers were obtained from all samples. All patients inboth groups had a fresh embryo transfer on day 3. The comparison betweenthe 2 groups reveals significant differences for implantation andongoing pregnancy rates/pick up (40.0% vs. 26.7% and 70.0% vs. 46.7;p<0.05, respectively). We noted 5 twin pregnancies in group 1 versus 0in the group 2 used as control. In addition, we observed that there wasno relationship between morphological aspects and the CC gene expressionprofile. On the basis of the analysis of 267 CC samples, we noted 27% ofCCs express genes which predict for embryos able to achieve pregnancy,42% of CCs did not, 31% of CCs showing gene expression for early arrestof embryo development.

Further, data generated from each oocyte (fertilization, embryodevelopment, transfer and pregnancy etc.) were recorded by anembryologist. All patients had two fresh embryos transfer on day 3.Pregnancy was confirmed by the presence of a fetal heartbeat byultrasound at 6-8 weeks.

CC samples were divided into 3 groups according embryos outcomes. Thefirst group (100% implantation) contained CCs from oocytes that produceda transferred embryo with successful twin pregnancy (n=10). The secondgroup (no pregnancy group) contained CCs from oocytes that resulted in atransferred embryo unable to implant (n=26), group 3 (other: freezingand early embryos arrest) contained CCs from oocytes that producedembryos presenting early arrest before the eight-cell stage or embryoscryopreserved (n=201) and group 4 (50% implantation rate).

The Characteristics of Cumulus Cells Samples

30 patients (age <36 years; ICSI) 267 CCs Positive group negative groupsGroup 1 Group 2 Group 3 Group 4 (100% (0% (other: freezing, (50%implantation implantation early embryos implantation rate) rate) arrest)rate) CCs nbr 10 26 201 30 CCs: Cumulus cells; ICSI: intracytoplasmicsperm injection

Quantitative RT-PCR analysis was performed to measure the relativeabundance of the transcripts of genes in CCs and expression data for all45 genes were obtained from 267 CC samples issued from 30 patients. Foreach gene, the relative intensity was calculated by dividing theintensity by the average intensity for each patient and for each gene.

Then, a SAM (Significance Analysis of Microarray) was realized to getthe more significant genes (p-value <0.05) that distinguish negativegroups who didn't have implantation or with early embryo arrest andpositive group who got a twin implantation.

Based on these results, 10 genes expression profiles appear to beparticularly reliable to predict different clinical conditions: (B)embryo unable to implant and (C) early embryo arrest. These candidatebiomarkers are listed in the below Table C.

TABLE C Clinical conditions predicted by the set of predictive genesGene Gene Symbol Gene name ID B: genes whose overexpressions arepredictive of embryos unable to implant PLA2G5 phospholipase A2, group V5322 GPC6 glypican 6 10082 ATF3 activating transcription factor 3 467SIAT6 ST3 beta-galactoside alpha-2,3-sialyltransferase 3 6487 PRKACAprotein kinase, cAMP-dependent, catalytic, alpha 5566 SLC40A1 solutecarrier family 40 (iron-regulated 30061 transporter), member 1 WNT6wingless-type MMTV integration site family, 7475 member 6 C: genes whoseoverexpressions are predictive of early embryo arrest NFIC nuclearfactor I/C (CCAAT-binding transcription 4782 factor) RBMS1 RNA bindingmotif, single stranded interacting 5937 protein 1 G0S2 G0/G1 switch 250486

These biomarkers in cumulus cells were able to predict differentclinical conditions. For example, overexpression of PLAG2G5 or GPC6 hasbeen found to be predictive of embryos unable to implant. On contrary,overexpression of NFIC or RBMS1 has been found to be predictive of anearly embryo arrest.

In the following step, different box plots (FIG. 1) were achieved forthose 10 genes of interest and a superior and an inferior cut-off foreach gene were determined, comparing patients with failed implantationor early embryo arrest to patients with twin implantation. When therelative intensity is superior of cut-off, the genes are supposed todiscriminate embryos unable to implant or early embryos arrest and thosewith twin pregnancy.

CONCLUSION

In most mammalian species including human, the cumulus cells whichsurrounds the oocyte are still present at the time of fertilization inthe oviduct and remain until embryonic implantation. Extracellularmatrix remodelling within and around the cumulus probably plays a keyrole in both of these steps. In this respect, we identified 10 genesexpressed in cumulus cells that are biomarkers for embryo potential andpregnancy outcome. In our study, we demonstrated that genes expressionprofile of CC which surrounds oocyte correlated to different outcomes,allowing the identification of a specific expression signature ofembryos developing toward pregnancy. In conclusion, we found adifferential gene expression between human cumulus cells from oocytesresulting in different pregnancy outcome from patients referred for ICSIor IVF. Our results indicate that analysis of cumulus cells surroundingthe oocyte is a non-invasive approach for embryo selection. Typically CCcan be collected immediately after oocyte pick-up, the CC can beanalyzed with a genomic test (G-test) to assess the potential of theembryo, and the embryo can be then be selected for fresh replacementbased on the G-test results.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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1-3. (canceled)
 4. A method of implanting an embryo in a female undergoing in vitro fertilization, comprising the steps of: a) collecting at least one oocyte with its cumulus cells from said female; b) measuring, in said cumulus cells surrounding said oocyte, an expression level of each of the 10 genes ATF3, SIAT6, PRKACA, PLA2G5, GPC6, G0S2, RBMS1, NFIC, SLC40A1 and WNT6; c) comparing the expression level of each of the 10 genes in the cumulus cells with control expression levels of the 10 genes from cumulus cells associated with competent oocytes; d) assessing said oocyte as having a higher probability of being competent if a cumulus cell surrounding the oocyte does not overexpress any of the 10 genes when compared to cumulus cells associated with a competent oocytes; e) fertilizing said oocyte having a higher probability of being competent in vitro to generate an embryo; and f) implanting said embryo in said female.
 5. The method of claim 4, wherein said step of measuring is carried out by i) extracting total mRNA from said cumulus cells; and ii) amplifying said mRNA to form cDNA using primers that are specific for said mRNA.
 6. The method of claim 4, wherein said step of measuring is carried out with nucleic acid probes.
 7. A method of implanting an embryo in a female undergoing in vitro fertilization, comprising the steps of: a) collecting oocytes from said female; b) generating embryos from said oocytes by fertilizing said oocytes in vitro; c) measuring, in cumulus cells surrounding each of said embryos, an expression level of each of the 10 genes ATF3, SIAT6, PRKACA, PLA2G5, GPC6, G0S2, RBMS1, NFIC, SLC40A1 and WNT6; d) comparing the expression level of each of the 10 genes in the cumulus cells with control expression levels of the 10 genes from cumulus cells associated with competent oocytes; e) assessing an embryo as having a higher probability of being competent if a cumulus cell associated with the embryo does not overexpress any of the 10 genes when compared to cumulus cells associated with competent oocytes; and f) implanting said embryo having a higher probability of being competent in said female.
 8. The method of claim 7, wherein said step of measuring is carried out by i) extracting total mRNA from said cumulus cells; and ii) amplifying said mRNA to form cDNA using primers that are specific for said mRNA.
 9. The method of claim 7, wherein said step of measuring is carried out with nucleic acid probes. 